I. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns methods and compositions involving microRNA (miRNAs) molecules. Certain aspects of the invention include applications for miRNAs in diagnostics, therapeutics, and prognostics of cervical cancer.
II. Background
In 2001, several groups used a cloning method to isolate and identify a large group of “microRNAs” (miRNAs) from C. elegans, Drosophila, and humans (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Several hundred miRNAs have been identified in plants and animals—including humans—which do not appear to have endogenous siRNAs. Thus, while similar to siRNAs, miRNAs are distinct.
miRNAs thus far observed have been approximately 21-22 nucleotides in length, and they arise from longer precursors, which are transcribed from non-protein-encoding genes. See review of Carrington et al. (2003). The precursors form structures that fold back on themselves in self-complementary regions; they are then processed by the nuclease Dicer (in animals) or DCL1 (in plants) to generate the short double-stranded miRNA. One of the miRNA strands is incorporated into a complex of proteins and miRNA called the RNA-induced silencing complex (RISC). The miRNA guides the RISC complex to a target mRNA, which is then cleaved or translationally silenced, depending on the degree of sequence complementarity of the miRNA to its target mRNA. Currently, it is believed that perfect or nearly perfect complementarity leads to mRNA degradation, as is most commonly observed in plants. In contrast, imperfect base pairing, as is primarily found in animals, leads to translational silencing. However, recent data suggest additional complexity (Bagga et al., 2005; Lim et al., 2005), and mechanisms of gene silencing by miRNAs remain under intense study.
Recent studies have shown that expression levels of numerous miRNAs are associated with various cancers (reviewed in Esquela-Kerscher and Slack, 2006; Calin and Croce, 2006). miRNAs have also been implicated in regulating cell growth and cell and tissue differentiation—cellular processes that are associated with the development of cancer.
Cervical cancer is the second most common cause of cancer in women worldwide (Pisani et al., 2002; Parkin et al., 2005). About 470,000 new cases are diagnosed and approximately 230,000 women die of cervical cancer every year (Pisani et al., 1999). While the majority (˜80%) of these new cases and deaths occur in developing countries, it is estimated that approximately 3,700 women will die from invasive cervical cancer in the United States in 2007 (Jemal et al., 2007).
Epidemiological and molecular studies have demonstrated that human papillomaviruses (HPVs) are the etiological agents of the vast majority (99.7%) of cervical cancers and their intraepithelial precursors (Pisani, et al., 2002; Parkin et al., 2005; zur Hausen, 2002). Approximately, fifty HPV types infect the anogenital tract including the uterine cervix (Pisani, et al., 2002; Parkin et al., 2005). “High-risk” HPV types are associated with intraepithelial lesions that can progress into invasive carcinomas. Among these, HPV 16 and HPV 18 are associated with 50% and 20%, respectively, of cervical squamous cell carcinomas (Bosch and de Sanjose, 2002; zur Hausen, 2002; Clifford et al., 2003). Other high-risk HPV types (31, 33, 35, 39, and 45 among others) are found in 20-30% of cervical cancers (Bosch and de Sanjose, 2002; zur Hausen, 2002; Clifford et al., 2003). High-risk HPV types are also associated with 25% of head and neck tumors, in particular tumors of the mouth, tonsils, esophagus and larynx (Gillison et al., 2000; Rose et al., 2006).
Cytological examination of cervical smears with Papanicolaou staining (Pap smears) is the screening method universally accepted for early detection of cervical cancer and its precursors. Pap smear screening has been very effective in reducing cervical cancer incidence and mortality. Abnormal pap smear results include mild dysplasias referred to as low-grade squamous intraepithelial lesions (LSIL) and moderate to severe dysplasias referred to as high-grade squamous intraepithelial lesions (HSIL). A Pap smear with LSIL or HSIL indicates a need for further examination and possible treatment. In one study (ALTS Group, 2000), over 80% of LSILs were found to be positive for HPV; however, almost 50% of LSILs will regress to normal. HSILs are generally considered to be pre-cancerous in nature and indicate more aggressive treatment. In addition, as many as 3 million Pap smears are classified as inconclusive in the U.S. every year, and cervical cancer is still a significant public health problem. Pap smear screening is imperfect due, in part, to sampling and staining errors, resulting in false-negatives. It is estimated that ˜17% of cervical cancers develop in women with previous false-negative Pap smears. It is also estimated that approximately 9% of cervical cancers develop in women with previous true-negative Pap tests.
As an adjunct to cytology screening, The United States Food and Drug Administration has approved a nucleic acid hybridization test for HPV for all women over 30 years old and as triage for women with inconclusive Pap smears. However, several problems associated with this assay include variable assay sensitivity, the inability to genotype certain strains of HPV, and the inability to detect about half of the high risk HPV types associated with cervical cancer (Begeron et al., 2000; de Cremoux et al., 2006).
A need exists for additional diagnostic assays that can assess the condition of cervical tissue in general and accurately distinguish pre-cancerous or cancerous tissue from non-cancerous tissue in particular.